The Survival of Motor Neurons Protein Determines the Capacity for snRNP Assembly: Biochemical Deficiency in Spinal Muscular Atrophy

Reduction of the survival of motor neurons (SMN) protein levelscauses the motor neuron degenerative disease spinal muscularatrophy, the severity of which correlates with the extent ofreduction in SMN. SMN, together with Gemins 2 to 7, forms acomplex that functions in the assembly of small nuclear ribonucleoproteinparticles (snRNPs). Complete depletion of the SMN complex fromcell extracts abolishes snRNP assembly, the formation of heptamericSm cores on snRNAs. However, what effect, if any, reductionof SMN protein levels, as occurs in spinal muscular atrophypatients, has on the capacity of cells to produce snRNPs isnot known. To address this, we developed a sensitive and quantitativeassay for snRNP assembly, the formation of high-salt- and heparin-resistantstable Sm cores, that is strictly dependent on the SMN complex.We show that the extent of Sm core assembly is directly proportionalto the amount of SMN protein in cell extracts. Consistent withthis, pulse-labeling experiments demonstrate a significant reductionin the rate of snRNP biogenesis in low-SMN cells. Furthermore,extracts of cells from spinal muscular atrophy patients havea lower capacity for snRNP assembly that corresponds directlyto the reduced amount of SMN. Thus, SMN determines the capacityfor snRNP biogenesis, and our findings provide evidence fora measurable deficiency in a biochemical activity in cells frompatients with spinal muscular atrophy.

INTRODUCTION

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Introduction

The process of pre-mRNA splicing is carried out by a macromolecularcomplex, the spliceosome, the major components of which arethe U1, U2, U5, and U4/U6 small nuclear ribonucleoprotein particles(snRNPs) (18, 34, 47). Each of the snRNPs (except for U6) iscomposed of one snRNA molecule, a set of seven common proteins,and several proteins that are specific to individual snRNAs(18, 27, 28, 47). SnRNP biogenesis begins with the transcriptionof the snRNAs in the nucleus followed by their nuclear exportto the cytoplasm, where the major assembly process of the snRNPstakes place. The common proteins, called Sm proteins, B/B',D1, D2, D3, E, F, and G, are arranged into a stable heptamericring, the Sm core, on a uridine-rich sequence motif, the Smsite, of the snRNAs (1, 2, 19, 41). The assembly of Sm coresis required for the subsequent modification of the 7-methylguanosine cap of snRNAs into a 2,2,7-trimethyl guanosine capas well as for the stability and function of the snRNPs (30,38). Properly assembled and modified snRNPs are then importedinto the nucleus, where additional snRNP-specific proteins associateto form fully functional snRNPs (10, 11, 13, 30, 31, 47).

Earlier studies have shown that snRNP assembly readily occursin vitro with purified total snRNP proteins (TPs) and snRNAsin an ATP-independent manner and without requirement for non-snRNPproteins (39, 40, 43). However, reconstitution of snRNPs inextracts from Xenopus laevis eggs and mammalian cells requiresATP (21, 32, 33, 37, 44), suggesting that snRNP assembly mightbe regulated by additional factors in vivo. Studies on a macromolecularcomplex containing the survival of motor neurons (SMN) proteinindicated that the SMN complex is required for the ATP-dependentsnRNP assembly (3, 9, 32, 33, 36, 37, 49). SMN is the proteinproduct of the gene responsible for spinal muscular atrophy(SMA), a common and often fatal genetic disorder in which motorneurons in the spinal cord degenerate (6, 8, 15, 22). Basedon the age of onset and the severity of the disease, SMA isclinically classified into three types: the severe type I, themoderate type II, and the mild type III. Studies on SMA patient-derivedcell lines have shown that the severity of SMA clinical phenotypesis closely linked to the degree of reduction of SMN proteinlevels (7, 23).

Immunodepletion or antibody inhibition of the SMN complex invitro demonstrated that the SMN complex is required for snRNPassembly (32, 33, 37). However, how much the SMN protein aswell as individual Gemins contribute to snRNP assembly and whathappens in SMA patients' cells, where the amount of SMN proteinis reduced to various degrees, have not been determined. Currentmethods using gel mobility shift assay to monitor snRNP assemblyare not suitable for quantitative analysis, due to the heterodispersemigration of large RNP complexes on native gels. To assess therelationship between the amount of SMN and the activity of Smcore assembly in cells and to facilitate further studies onthe mechanism of SMN complex function, we developed a sensitiveand quantitative assay for snRNP assembly.

The assay is based on the isolation of high-salt- and heparin-resistantSm cores formed on biotin-labeled snRNAs with magnetic beadsbearing anti-Sm antibodies. The amount of snRNPs assembled inthe reaction is then determined by luminescence detection ofthe biotin molecules on the snRNAs. Importantly, we show thatthe extent of assembly is directly dependent on the amount ofthe SMN complex in cell extracts and that extracts from cellsof SMA patients have a lower capacity for snRNP assembly, proportionalto the reduction of SMN protein in these cells. In vivo pulse-labelingexperiments demonstrate that the rate of biogenesis of the majorsnRNPs is strongly reduced in cells with low SMN. These findingsprovide evidence for a deficiency in a specific biochemicalactivity in SMA patients and present a powerful method for studyingthe activity of the SMN complex.

MATERIALS AND METHODS

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Materials and Methods

In vitro transcription of RNAs. Plasmids for in vitro transcription of snRNAs were describedelsewhere (10, 13, 16, 30). For radiolabeling of RNAs, in vitrotranscription was carried out in the presence of [³²P]UTP aspreviously described (48). Biotin-labeled RNAs were producedaccording to the suggestions of the manufacturer (Ambion) withthe modification that 5 mM biotin-UTP (Roche) and 2.5 mM UTPwere present. All of the labeled RNAs were purified by electrophoresison 7 M urea-6% polyacrylamide gels, precipitated with ethanol,and resuspended in nuclease-free water. The concentrations ofthe biotin-labeled RNAs were determined by absorbance at 260nm.

Cell lines and cell culture maintenance. The maintenance of the S5 cell line, which is a chicken DT40cell line with targeted disruption of the SMN gene, has beendescribed previously (45). Epstein-Barr virus-transformed lymphoblastcell lines derived from a 6-month-old SMA type I patient (GM10684)and an age- and gender-matched individual with a syndrome unrelatedto SMA as a control (GM12497) were obtained from Coriell CellRepositories and maintained in RPMI 1640 medium (Gibco BRL)containing 10% fetal bovine serum (HyClone) and 1% penicillin-streptomycin(Gibco BRL). Primary fibroblast cell lines from four SMA typeI patients (GM00232, GM09677, GM03813, and GM03815), one heterozygouscarrier (GM03814), and two apparently healthy controls (GM08333and GM00498) were also obtained from Coriell Cell Repositories.They were maintained in minimal essential medium (Gibco BRL)containing 15% fetal bovine serum, 2 mM L-glutamine, and 1%penicillin-streptomycin.

Preparation of cytoplasmic extracts from cultured cells. For each sample in the assays shown, the same number ( ${approx}$ 4 x 10⁷)of cells were harvested and washed twice with phosphate-bufferedsaline. For the SMA fibroblast cell lines, cells were used atan early and identical passage stage. Cytoplasmic extracts competentfor snRNP assembly were prepared as described previously (37).The protein concentrations of the various extracts were determinedusing the Bio-Rad protein assay (Bio-Rad). All of the extractswere adjusted to the same final protein concentration ( ${approx}$ 15 mg/mlfor extracts prepared from HeLa cells, S5 cells, or SMA lymphoblastoidcells, and 3 mg/ml for extracts of SMA fibroblast cells) andwere quickly frozen in liquid nitrogen and stored in aliquotsat –80°C.

Assay for in vitro assembly of snRNPs. For in vitro Sm core assembly on ³²P-labeled snRNAs in cytoplasmicextracts, the reactions were carried out at 30°C for 1 husing standard assembly reaction conditions (37). Subsequently,half of the reaction mixtures was loaded onto native gels forelectrophoretic mobility shift assays as described previously(37). The other half of the samples was immunoprecipitated withthe anti-Sm monoclonal antibody Y12 (24), and the immunoprecipitatedRNAs were isolated and analyzed by electrophoresis on 7 M urea-8%polyacrylamide gels.

For quantitative in vitro assembly assays on magnetic beads,cytoplasmic extracts were prepared and used for assembly onbiotin-labeled snRNAs using the standard reconstitution conditionsin 96-well plates. Following the reactions, Y12 antibodies immobilizedonto the magnetizable Dynabeads protein A (Dynal Biotech ASA,Oslo, Norway) in 100 µl of RSB-500 buffer (10 mM Tris-HCl,pH 7.5, 500 mM NaCl, 2.5 mM MgCl₂) containing 2 mg/ml heparin,0.1% NP-40, and 0.2 U/µl RNasin RNase inhibitor (Promega)were added to each well. Immunoprecipitations in the 96-wellplates were carried out with gentle mixing at 750 rpm in a Thermomixer(Eppendorf, Germany) at 30°C for 1 h. The plates were subsequentlytransferred to a Kingfisher 96 magnetic particle processor (ThermoLabsystems, Vantaa, Finland) for automatic washing of the Dynabeadsin each well with 200 µl of wash buffer (RSB-500, 0.1%NP-40) five times. After the last wash, the beads that werebound with Y12-immunoprecipitated snRNPs were then resuspendedin 120 µl of wash buffer containing 0.08 µg/ml horseradishperoxidase-conjugated NeutrAvidin (Pierce). Following incubationat 30°C for 1 h with gentle mixing, the beads in each wellwere again washed five times by the Kingfisher 96 magnetic particleprocessor and finally resuspended in 150 µl of SuperSignalenzyme-linked immunosorbent assay (ELISA) Femto substrate workingsolution (Pierce). The plates were transferred to a Wallac Victor2multilabel plate reader (Perkin-Elmer) for luminescence measurementsat 495 nm. The resulting data were analyzed with Microsoft Excel.

Antibodies and quantitative immunoblotting. The anti-SMN (62E7), anti-Gemin2 (2E17), anti-Gemin3 (12H12),anti-Gemin4 (17D10), anti-Gemin5 (10G11), anti-Y14 (1F12), andanti-Sm (Y12) monoclonal antibodies have been described previously(4, 5, 12, 20, 24-26). The anti-mouse immunoglobulin G secondaryantibody labeled with IRDye800 (Rockland) was used at 1:5,000.Proteins from 20 µg of cytoplasmic extracts were separatedon NuPAGE 4 to 12% Bis-Tris gels (Invitrogen) and transferredto nitrocellulose membranes. Quantitative immunoblotting wasperformed as suggested by the manufacturer (Li-Cor). The membraneswere scanned on an Odyssey infrared imaging system (Li-Cor),and the intensity of the protein bands was analyzed using thesoftware provided by the manufacturer.

Pulse-label measurements of the rate of snRNP biogenesis in cells. Three days after chicken S5 cells were cultured in medium containing10 or 18 ng/ml tetracycline, equal numbers of cells (10⁷) werepulse-labeled with 25 µCi/ml [³H]uridine (Amersham) forone hour. Total RNAs were isolated from 10% of the labeled cellsusing TRIzol reagent (Invitrogen). Total cell extracts fromthe remaining cells were subjected to immunoprecipitation byY12. The immunoprecipitated RNAs were isolated by proteinaseK treatment followed by phenol-chloroform extraction and ethanolprecipitation. Both total RNAs and Y12-immunoprecipitated RNAswere analyzed by electrophoresis on 7 M urea-8% polyacrylamidegels. Gels were then treated with Amplify solution (Amersham)and dried for autoradiography.

Analysis of steady-state snRNP levels in cells. To determine the overall amount of snRNPs at steady state incells, Y12 immunoprecipitations were performed using cell extractsfrom 10⁷ S5 cells that were grown in the presence of either10 or 18 ng/ml tetracycline. Y12 immunoprecipitated RNAs wereisolated and radioactively labeled at the 3' end with [ ${alpha}$ -³²P]pCp(Perkin-Elmer) and T4 RNA ligase (New England Biolabs). Thelabeled RNAs were resolved by electrophoresis on 7 M urea/8%polyacrylamide gels and analyzed by autoradiography.

RESULTS

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Results

Development of a quantitative assay for snRNP assembly. Assembled Sm cores comprised of seven-membered rings of Sm proteins,unlike the complexes of individual Sm proteins or a subset ofSm proteins with RNA, are extremely sturdy and resist dissociationeven in the presence of high salt, heparin, and urea (14, 16,40). This property provides the basis for a gel mobility shiftassay to assess Sm core formation. In these assays, ³²P-labeledsnRNAs are incubated with purified total snRNP proteins or acell fraction containing Sm proteins, such as extracts fromHeLa cells or Xenopus laevis eggs, and the stable Sm cores,resolved by electrophoresis from free RNAs, are visualized bytheir characteristic mobility shift after autoradiography ofthe native polyacrylamide gels (21, 32, 33, 37, 39, 44).

As has been shown previously, using this gel shift assay, Smcores formed on Sm site-containing U1, U4, and U5 snRNAs whenincubated in HeLa cytoplasmic extracts (Fig. 1A). Sm core assemblywas strictly dependent on Sm proteins supplied by the extracts(Fig. 1A, +CE) and on the presence of an Sm site on the snRNAs(Fig. 1A, ${Delta}$ Sm). Sm cores assembled only inefficiently on U1A3(Fig. 1A), a U1 snRNA mutant that has three nucleotide substitutionsin the loop of stem-loop 1 and is impaired in binding to theSMN complex (48). However, due to the heterodisperse migrationof large RNP complexes on native gels, the gel shift assay isnot a sensitive and objective method to accurately quantitateSm core assembly.

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FIG. 1. Analysis of in vitro-assembled snRNPs by the mobility gel shift assay and by the magnetic beads assay. (A) [³²P]UTP-labeled U1, U1 ${Delta}$ Sm, U1A3, U4, U4 ${Delta}$ Sm, U5, or U5 ${Delta}$ Sm snRNA was mixed with HeLa cytoplasmic extracts (CE) containing 25 µg total proteins (+ lanes) or with buffer only (– lanes) for in vitro assembly of Sm cores. The reaction mixtures were analyzed by electrophoresis on 6% native polyacrylamide gels. The brackets on the right indicate the positions of assembled Sm cores and the free RNAs. The complex that results from the binding of the U1-specific protein U1A to stem-loop 2 of the U1 snRNA is marked on the left. The band indicated by an asterisk is likely to be U1A/Sm core complexes. (B) The same reaction mixtures as used in panel A were immunoprecipitated by Y12 bound to protein A-Sepharose beads. RNAs were isolated from the bound fractions and analyzed by electrophoresis on 7 M urea-8% polyacrylamide gels. The RNAs that migrate at different positions on the gel are indicated on the left. (C) SnRNAs were produced by in vitro transcription in the presence of biotin-UTP instead of [³²P]UTP, and similar assembly reactions as in A were carried out with these biotinylated RNAs or without any RNA using either HeLa cytoplasmic extracts (+ lanes) or buffer (– lanes). The amount of the Sm cores assembled on these RNAs was assessed by the magnetic beads assay as depicted in Fig. 2. The error bars, which for some data points are too small to be seen on the figure, represent standard deviations from three independent experiments.

To further confirm the identity of the assembled complexes andto assess snRNP assembly more accurately, the assembly reactionmixtures were subjected to immunoprecipitations with the anti-Smantibody Y12 (24) under stringent conditions such that onlysnRNAs that have assembled high-salt- and heparin-resistantSm cores could be immunoprecipitated. Following Y12 immunoprecipitation,³²P-labeled snRNAs on which Sm cores formed were eluted andanalyzed by urea-polyacrylamide gel electrophoresis (14, 16,37). Similar to the gel shift assay, Sm cores assembled on thewild-type snRNAs but not on the respective ${Delta}$ Sm mutants (Fig.1B). A weak signal was detected for the U1A3 mutant, demonstratingmore clearly than on the gel shift assay a low level of Sm coreformation on this mutant (Fig. 1B).

The level of snRNP assembly can be estimated by autoradiographyon a phosphorimager. While the ³²P-labeled RNA bands obtainedafter immunoprecipitation with anti-Sm antibodies are more suitableto estimate assembled Sm cores, this approach, using radiolabeledRNAs, still has considerable shortcomings, such as the shorthalf-life of the ³²P-labeled RNAs and the laborious handlingof the hazardous materials.

To facilitate the study of the snRNP assembly process, we developedan assay that allows quantitative measurement of Sm core formation(Fig. 2). Instead of labeling RNAs with [³²P]UTP, snRNAs wereprepared by in vitro transcription in the presence of biotin-UTP.Following in vitro assembly reactions with the biotin-labeledRNAs, immunoprecipitations of the Sm cores were carried outunder stringent conditions, including high salt (500 mM NaCl)and heparin (2 mg/ml). The immunoprecipitations were carriedout with Y12 antibodies immobilized on magnetic beads in a 96-wellplate format, which allows automatic cycles of washing and mixingof the beads on a robotic manifold. Subsequently, horseradishperoxidase-conjugated avidin, which binds tightly to biotin,was used to recognize the biotinylated RNAs in the Y12 immunoprecipitatedSm cores. This step also serves to amplify the signals for theluminescence measurement of the horseradish peroxidase activityon an automatic plate reader.

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FIG. 2. Schematic depiction of the magnetic bead assay procedure for the detection of in vitro-assembled snRNPs.

The results obtained using this assay system (Fig. 1C) stronglyresemble those of Fig. 1A and 1B. The signal indicating theamount of assembly on U1, U4, and U5 is more than 100-fold abovebackground (Fig. 1C, -RNA, -CE). Readings on all ${Delta}$ Sm mutantswere close to background, demonstrating the strict dependenceof assembly on an Sm site. In this assay, the assembly of Smcores on U1A3 is more significant than what was observed bythe gel shift assay or Y12 immunoprecipitation with ³²P-labeledsnRNAs. Since U1A3 binds to the SMN complex with lower affinitythan wild-type U1 (data not shown) and Sm cores assemble onU1A3 with slower kinetics in vivo in Xenopus oocytes (48), thedifference might be due to the higher concentration of U1A3RNA and longer reaction time used in this assay. As indicatedby the error bars in Fig. 1C, the experimental variation inindependent experiments is typically less than 5% of the signal.This assay for snRNP assembly is more sensitive, much less labor-intensivethan previous methods, and amendable to high-throughput automation.

While the assembly of Sm cores from TPs is ATP independent,energy is required for this process to occur in cell extracts(21, 32, 33, 37, 44). As shown in Fig. 3A, Sm core assemblyin extracts requires the addition of exogenous ATP and doesnot occur with adenosine 5'-(ß, ${gamma}$ -imido)triphosphate(AMP-PNP), a nonhydrolysable ATP analog, suggesting that ATPhydrolysis is required for assembly. To optimize the assay conditions,we examined the effect of several parameters on the Sm coreassembly. Assembly on wild-type snRNA depends on RNA concentration,the amount of cytoplasmic extracts, and the reaction time (Fig.3B to D). In contrast, Sm cores do not form on ${Delta}$ Sm mutant snRNAseven with excess RNAs, extracts, and reaction time (Fig. 3B to D).Profiles similar to those presented in Fig. 3 for U4were obtained for U1 and U5 (data not shown). Since assemblyactivity corresponds linearly to the amount of extract (Fig.3C) and plateaus at higher RNA concentration and longer reactiontime (Fig. 3B and 3D), we set the parameters used for our standardassembly reaction at 5 nM biotinylated snRNA, cytoplasmic extractscontaining 25 µg total proteins, and a reaction time of60 min.

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FIG. 3. Specific parameters that affect snRNP assembly in vitro. In vitro assembly reactions were performed using standard conditions except one of the following parameters. Sm core assembly on U4 (A, B, C, and D) and U4 ${Delta}$ Sm (B, C, and D) was examined by the magnetic beads assay and graphed against ATP or AMP-PNP concentration (A), the snRNA concentration (B), the amount of HeLa cytoplasmic extract (C), or the assembly reaction time (D). Error bars, which are too small to be visible for some of the data points, represent standard deviations from two independent experiments.

SMN protein determines the capacity for snRNP assembly. It has been previously shown that the assembly of Sm cores isabolished when SMN complexes are depleted in vitro from cellextracts (32, 37). Since the assembly activity correlates withthe amount of cytoplasmic extract (Fig. 3C), we asked whetherit is the amount of SMN in the extract that determines snRNPassembly capacity. To address this, SMN expression was reducedin HeLa cells by RNA interference (RNAi) and this resulted ina drastic reduction in the assembly activity of the extractsprepared from these cells (Feng and Dreyfuss, personal communication).However, RNAi cannot be regulated to obtain a desired levelof reduction in the amount of the protein. We therefore usedthe S5 cell line, derived from chicken DT40 cells, in whichthe endogenous SMN gene is disrupted by homologous recombinationand SMN protein is exogenously expressed from a tetracycline-repressiblepromoter (45). This allows SMN expression to be tightly controlledto various levels by adjusting the concentration of tetracyclinein the culture media (45). Since the growth of S5 cells is arrestedafter 72 h in the presence of 18 ng/ml tetracycline and higherconcentrations of tetracycline lead to cell death (45), we analyzedS5 cells which were normally maintained in 10 ng/ml tetracyclineand then were split into culture media containing 10, 12, 14,16, or 18 ng/ml tetracycline for 3 days. Equal numbers of cells( ${approx}$ 4 x 10⁷) were harvested for each of the tetracycline concentrationsand used to prepare cytoplasmic extracts.

The expression level of Y14, a component of the exon-junctioncomplex (20) and unrelated to the snRNP assembly process, remainedlargely unchanged at various tetracycline concentrations (Fig.4A) and was therefore used as an internal control to determinethe relative protein levels of SMN and Sm proteins B/B'. Asshown in Fig. 4B, the level of the SMN protein decreased withincreasing tetracycline concentration. Cells cultured at 18ng/ml tetracycline contained about 30% SMN compared to thoseat 10 ng/ml tetracycline. Unlike the reduction of SMN, SmB/B'protein levels were not significantly changed.

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FIG. 4. Reduced snRNP assembly correlates with decreased SMN protein levels in extracts from chicken S5 cells. (A) Cytoplasmic extracts were prepared from S5 cells cultured at 10, 12, 14, 16, or 18 ng/ml tetracycline. SMN, SmB/B', and Y14 proteins in each of these extracts containing 20 µg total proteins were detected by quantitative Li-Cor Western blotting analysis. (B) The intensity of each protein band in panel A was analyzed using the Odyssey infrared imaging system. SMN and SmB/B' proteins levels in extracts of various tetracycline concentrations were normalized using the Y14 signals as an internal control. The relative SMN and SmB/B' protein levels at 18 ng/ml tetracycline were set to 1. The relative protein levels at other tetracycline concentrations were calculated as ratios to those at 18 ng/ml tetracycline. (C) SnRNP assembly capacities of the same extracts as in A on U4 snRNA were examined by the magnetic bead assay. The relative assembly activity of extract at 18 ng/ml tetracycline was set to 1. The relative assembly activities of the other extracts were calculated as ratios to that at 18 ng/ml tetracycline. (D) The relative assembly activity at each tetracycline concentration as in panel C was plotted on the y axis against the corresponding relative SMN protein level as in panel B on the x axis. The data points fitted best to a linear graph (R² = 0.9911).

We next employed the magnetic beads assay to quantitate theassembly activity of these cell extracts. The capacity for assemblyof Sm cores decreased with the reduction in SMN protein (Fig.4C). When the relative assembly activity at each tetracyclineconcentration was plotted against the corresponding relativeSMN protein level, the data points fitted best to a linear graphwith a significant R² value (R² = 0.9911), indicating a directcorrelation between the SMN protein level and assembly activity(Fig. 4D). These results indicate that the SMN protein determinesthe capacity of Sm core assembly in cell extracts even if theycontain similar, nonlimiting amounts of Sm proteins.

Cells with low SMN accumulate snRNPs more slowly. To determine if the reduced snRNP assembly capacity observedin extracts of cells with low SMN is reflected in vivo, we measuredthe rate of snRNP biogenesis in S5 cells cultured in the presenceof either 10 ng/ml tetracycline (normal SMN) or 18 ng/ml tetracycline(low SMN) by pulse-labeling. Pulse-labeling of these cells showedthat the rate of accumulation of the major snRNAs and snRNPs,determined by one hour of [³H]uridine incorporation into themajor Sm core-containing snRNAs, particularly U1 and U2, wasdrastically reduced (Fig. 5A, Y12 IP RNA). Albeit to a lesserextent, the total amount of labeled U1 and U2 snRNAs, as wellas other RNAs (e.g., 5S and tRNA) produced in the same timeinterval was also reduced (Fig. 5A, Total RNA).

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FIG. 5. Cells with low SMN accumulate snRNPs more slowly. (A) To determine the rate of snRNP biogenesis in vivo, S5 cells cultured at 10 or 18 ng/ml tetracycline were pulse-labeled with 25 µCi/ml [³H]uridine for one hour. Total RNAs from 10% of the labeled cells and snRNAs isolated by immunoprecipitations with Y12 from the remaining cells were analyzed on 7 M urea-8% polyacrylamide gel. Known RNAs are indicated on the left. (B) To measure the level of snRNPs in vivo at steady state, Y12 immunoprecipitations were performed on S5 cells cultured at 10 or 18 ng/ml tetracycline. The immunoprecipitated RNAs were isolated, radioactively labeled at the 3' end, and analyzed on 7 M urea-8% polyacrylamide gel. Known RNAs are indicated on the left.

It is possible that low-SMN cells also have reduced transcriptionrates and/or that RNAs, especially snRNAs, that are not assembledare rapidly degraded (29). The possibility that the rate oftranscription is reduced is consistent with previous observationssuggesting a role for the SMN complex in the formation of transcriptionfactories (35). We conclude that SMN is a critical factor indetermining the rate of production of snRNPs in cells. We notethat the overall steady-state amount of the major snRNAs andsnRNPs, determined by 3'-end labeling of the Y12 immunoprecipitatedRNAs (Fig. 5B), was not significantly different in normal- andlow-SMN cells. However, the low-SMN cells have a much slowergrowth rate than the normal-SMN cells, possibly to allow thesecells to accumulate the necessary amount of RNPs, which nowoccurs at a slower rate.

Deficiency in snRNP assembly in cell extracts of SMA patients. In light of the findings described above that SMN protein levelsparallel the capacity of snRNP assembly in HeLa cells and inchicken S5 cells, we asked whether cells from SMA patients werealso deficient in snRNP assembly activity. We first examineda lymphoblastoid cell line derived from a type I SMA patient(GM10684), using a similarly established cell line from an age-and gender-matched individual as a control (GM12497). Immunoblottingshowed that SMN in the patient cells was considerably reduced,whereas the levels of Gemins 2 to 5 and SmB/B' were similarto the control (Fig. 6A). Compared to the control, the snRNPassembly activity of the patient cell extract was 48% ±10% in three independent experiments. This assembly capacitycorrelates well with the SMN level, which was 38% ± 6.5%of the control (Fig. 6B), demonstrating a biochemical deficiencyin the cells of an SMA patient.

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FIG. 6. Cells of SMA patients are deficient in their capacity for snRNP assembly. (A) Cytoplasmic extracts were prepared from two lymphoblast cell lines. One was established from an SMA type I patient (GM10684) and the other from an age- and gender-matched individual (GM12497) as a control. SMN, Gemins 2 to 5, and SmB/B' proteins were detected by quantitative Li-Cor Western blotting analysis, using Y14 as a loading control. (B) The intensity of each of the protein bands in panel A was analyzed using the Odyssey infrared imaging system. The SMN protein level in each of the cell extract was normalized using Y14 signal as an internal control. The relative level of SMN protein in the cell extract of an SMA patient (GM10684) was calculated as the percentage of that of the control (GM12497). Similarly, the relative assembly activity in cell extracts of the patient (GM10684) was calculated as a percentage of the control (GM12497). (C) Cytoplasmic extracts from SMA patient fibroblast cell lines were assayed by the magnetic bead assay. SnRNP assembly activities in cell extracts of the controls (GM00498, GM08333, and GM03814) were set to be 100%. Assembly activities in cell extracts of the SMA type I patients (GM09677, GM00232, GM03813, and GM03815) were expressed as percentages of their respective controls.

We further studied several available sets of primary fibroblastcell lines derived from type I SMA patients or heterozygouscarriers, as well as unaffected individuals (Table 1). The full-lengthSMN transcript levels and SMN protein levels in patient cells(GM09677, GM00232, and GM03813) were estimated to be about 40to 50% of those of the respective controls (GM00498, GM08333,and GM03814) (42). However, we observed that the overall SMNprotein levels in these fibroblast cells are very low and passagedependent (data not shown). Consequently, the assembly activitiesin these cells were considerably lower than those in the lymphoblastoidcells.

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TABLE 1. Fibroblast cell lines derived from SMA patients and control individuals^a

The assembly assay revealed that extracts from the patient fibroblastcells had approximately 40% of the assembly activity of thecorresponding controls (Fig. 6C), consistent with the degreeof reduction of SMN protein in these patient cells. Unexpectedly,GM03815 cells, described in the Coriell Cell Repository as havingbeen obtained from the heterozygous carrier father of the donorof the GM03813 cells, also showed a similar level of deficiencyin snRNP assembly (Fig. 6C). Consistent with this, genetic linkageanalysis indicated that GM03815 is, in fact, a male siblingof GM03813 (R. Wilson, personal communication). Indeed, anotheraffected son was listed in the family tree. These observationsdemonstrate that as a result of reduced levels of SMN proteinexpression, cells of SMA patients are deficient in snRNP assemblyin vitro and suggest that the snRNP assembly assay can be usedas a potential diagnostic tool for SMA.

DISCUSSION

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Discussion

Previous experiments have demonstrated that the SMN complexis required for snRNP assembly. Specifically, complete or nearlycomplete removal or inhibition of the SMN complex results inthe inhibition of Sm core assembly in vitro (32, 37). Here,using an in vitro assay we developed for the quantitative measurementof the Sm core assembly process in cell extracts, we show thatthere is a linear correlation between the amount of SMN presentin the cell extract and the amount of Sm cores that can be formedon specific RNA substrates, suggesting that the amount of SMNdetermines the capacity for Sm core assembly. SMA results froma reduction in the amount of the full-length SMN protein (6,8, 15, 22). Studies on a collection of SMA patient cell linesrevealed that SMN expression is more reduced in the severe form(type I) than the mild form (type III) of the disease, demonstratinga direct correlation between the degree of reduction of SMNprotein levels in SMA patients and the severity of their clinicalphenotypes (7, 23). However, an understanding of the molecularconsequences of the reduced levels of SMN in patients' cellshas been lacking.

Our finding that snRNP assembly is impaired in cells of SMApatients provides the first direct evidence for a biochemicaldeficiency, namely, reduced capacity to assemble Sm cores, inSMA patients. Due to a limited availability of SMA patient cells,our studies focused on the severe type I SMA patients. However,the correlation we find is strong, exhibiting direct proportionalitybetween the amount of the SMN protein and assembly capacityin vitro. Consistent with the reduced activity observed in extractsof cells with low SMN, the rate of production of snRNPs in thesecells is profoundly reduced.

Although the overall amount of the major snRNPs in the samecells is not reduced, the strong deficiency in their rate ofaccumulation could be detrimental to cells in several ways.For dividing cells, biosynthetic capacity, such as the overalltranslation rate, has been linked to the control of cell growthand division (17, 46). Similarly, the slower rate of snRNP accumulationcould cause a delay in cell cycle progression. This is indeedthe case for S5 cells where the growth rate slows down proportionallyto the reduction in SMN (45). In nondividing cells, such asmotor neurons, an unmet demand for timely snRNP production ata particular point in the growth and development of the cellcould have severe consequences for the cell. It could lead toa deficit in functions that depend on an adequate amount ofthe major snRNPs (e.g., a general decrease in pre-mRNA splicingor an altered processing pattern of pre-mRNAs that are requiredby that cell), or to a deficit in a specific snRNP (e.g., anRNA of a lower abundance or one that has a lower affinity forthe SMN complex). It is also possible that reduced amounts ofthe SMN complex result in some loss of the regulation of Smcore assembly, leading to loss of fidelity of Sm core assembly,such that Sm cores assemble on RNAs that are not supposed toreceive them. This could be harmful to cells, as it may interferewith the normal functions of these RNAs or cause them to aggregate.We note that the experiments we carried out here examined onlythe formation of the major snRNPs, and it is therefore possiblethat the formation of minor snRNPs, motor neuron-specific snRNPs,or other RNAs that require the SMN complex for RNP assemblyis strongly affected by the reduction in SMN.

The method we describe here is sensitive, quantitative, easyto set up, and readily suitable for laboratory automation. Unlikethe methods commonly used so far, such as RNP gel shift andanti-Sm immunoprecipitation followed by gel electrophoresis,this assay does not require radioactive labeling of the RNAs,nor does it require gel electrophoresis. The biotinylated RNAsubstrates used for the assay can be prepared ahead of timeand stored for many months, allowing off-the-shelf and long-termuse. The assay should facilitate detailed studies of the mechanismsof action of the SMN complex and on the process of Sm core assembly.Combined with other approaches, such as RNA interference tosystematically reduce protein expression, it should now be possibleto determine the roles of the individual Gemins and other componentsof interest in the assembly reaction. The assay can also beused on extracts from patient cells. The sensitivity of theassay and its colinearity with the amount of SMN make it suitableas an additional measure for the characterization of SMA andas a means to identify potential modifiers, both genetic andpharmacological, of the disease phenotype.

ACKNOWLEDGMENTS

We are grateful to Joan A. Steitz for providing the Y12 monoclonalantibody. We thank Stephen A. Liebhaber, Tracey J. Golembe,and the members of our laboratory for stimulating discussionsand helpful comments on the manuscript. We are also gratefulto Gina Daly for secretarial assistance.

This work was supported by the Association FrançaiseContre les Myopathies (AFM) and by a grant from the NationalInstitutes of Health. G.D. is an Investigator of the HowardHughes Medical Institute.

FOOTNOTES

* Corresponding author. Mailing address: Howard Hughes Medical Institute, Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148. Phone: (215) 898-0398. Fax: (215) 573-2000. E-mail: gdreyfuss{at}hhmi.upenn.edu.

REFERENCES

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